Jolanda van Hengel

A novel role for p120 catenin in E-cadherin function.

Îndirect evidence suggests that p120-catenin (p120) can both positively and negatively affect cadherin adhesiveness. Here we show that the p120 gene is mutated in SW48 cells, and that the cadherin adhesion system is impaired as a direct consequence of p120 insufficiency. Restoring normal levels of p120 caused a striking reversion from poorly differentiated to cobblestone-like epithelial morphology, indicating a crucial role for p120 in reactivation of E-cadherin function. The rescue efficiency was enhanced by increased levels of p120, and reduced by the presence of the phosphorylation domain, a region previously postulated to confer negative regulation. Surprisingly, the rescue was associated with substantially increased levels of E-cadherin. E-cadherin mRNA levels were unaffected by p120 expression, but E-cadherin half-life was more than doubled. Direct p120–E-cadherin interaction was crucial, as p120 deletion analysis revealed a perfect correlation between E-cadherin binding and rescue of epithelial morphology. Interestingly, the epithelial morphology could also be rescued by forced expression of either WT E-cadherin or a p120-uncoupled mutant. Thus, the effects of uncoupling p120 from E-cadherin can be at least partially overcome by artificially maintaining high levels of cadherin expression. These data reveal a cooperative interaction between p120 and E-cadherin and a novel role for p120 that is likely indispensable in normal cells.

Implication of the MAGI-1b/PTEN signalosome in stabilization of adherens junctions and suppression of invasiveness

We recently established the critical role of the lipid phosphatase activity of the PTEN tumor suppressor in stabilizing cell‐cell contacts and suppressing invasiveness. To delineate the effector systems involved, we investigated the interaction of PTEN with E‐cadherin junctional complexes in kidney and colonic epithelial cell lines. PTEN and the p85 regulatory subunit of phosphatidylinositol 3‐OH kinase (PI3K) co‐immunoprecipitated with E‐cadherin and catenins. By using a yeast two‐hybrid assay, we demonstrated that PTEN interacted indirectly with β‐ catenin by binding the scaffolding protein MAGI‐1b. This model was corroborated in various ways in mammalian cells. Ectopic expression of MAGI‐1b potentiated the interaction of PTEN with junctional complexes, promoted E‐cadherin‐dependent cell‐cell aggregation, and reverted the Src‐induced invasiveness of kidney MDCKts‐src cells. In this model, MAGI‐1b slightly decreased the activity of AKT, a downstream effector of PI3K. By using dominant‐negative and constitutively active AKT expression vectors, we demonstrated that this kinase was included in the pathways involved in Src‐induced destabilization of junctional complexes and was necessary and sufficient to trigger invasiveness. We propose that the recruitment of PTEN at adherens junctions by MAGI‐1b and the local down‐regulation of phosphatidylinositol‐3,4,5‐trisphosphate pools and downstream effector systems at the site of cell‐cell contacts are focal points for restraining both disruption of junctional complexes and induction of tumor cell invasion.

Megakaryocyte-specific RhoA deficiency causes macrothrombocytopenia and defective platelet activation in hemostasis and thrombosis

Vascular injury initiates rapid platelet activation that is critical for hemostasis, but it also may cause thrombotic diseases, such as myocardial infarction or ischemic stroke. Reorganizations of the platelet cytoskeleton are crucial for platelet shape change and secretion and are thought to involve activation of the small GTPase RhoA. In this study, we analyzed the in vitro and in vivo consequences of megakaryocyte- and platelet-specific RhoA gene deletion in mice. We found a pronounced macrothrombocytopenia in RhoA-deficient mice, with platelet counts of approximately half that of wild-type controls. The mutant cells displayed an altered shape but only a moderately reduced life span. Shape change of RhoA-deficient platelets in response to G(13)-coupled agonists was abolished, and it was impaired in response to G(q) stimulation. Similarly, RhoA was required for efficient secretion of α and dense granules downstream of G(13) and G(q). Furthermore, RhoA was essential for integrin-mediated clot retraction but not for actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo, RhoA deficiency resulted in markedly prolonged tail bleeding times but also significant protection in different models of arterial thrombosis and in a model of ischemic stroke. Together, these results establish RhoA as an important regulator of platelet function in thrombosis and hemostasis.

The lipid phosphatase activity of PTEN is critical for stabilizing intercellular junctions and reverting invasiveness

To analyze the implication of PTEN in the control of tumor cell invasiveness, the canine kidney epithelial cell lines MDCKras-f and MDCKts-src, expressing activated Ras and a temperature-sensitive v-Src tyrosine kinase, respectively, were transfected with PTEN expression vectors. Likewise, the human PTEN-defective glioblastoma cell lines U87MG and U373MG, the melanoma cell line FM-45, and the prostate carcinoma cell line PC-3 were transfected. We demonstrate that ectopic expression of wild-type PTEN in MDCKts-src cells, but not expression of PTEN mutants deficient in either the lipid or both the lipid and protein phosphatase activities, reverted the morphological transformation, induced cell–cell aggregation, and suppressed the invasive phenotype in an E-cadherin–dependent manner. In contrast, overexpression of wild-type PTEN did not counteract Ras-induced invasiveness of MDCKras-f cells expressing low levels of E-cadherin. PTEN effects were not associated with marked changes in accumulation or phosphorylation levels of E-cadherin and associated catenins. Wild-type, but not mutant, PTEN also reverted the invasive phenotype of U87MG, U373MG, PC-3, and FM-45 cells. Interestingly, PTEN effects were mimicked by N-cadherin–neutralizing antibody in the glioblastoma cell lines. Our data confirm the differential activities of E- and N-cadherin on invasiveness and suggest that the lipid phosphatase activity of PTEN exerts a critical role in stabilizing junctional complexes and restraining invasiveness.

Mutations in the area composita protein αT-catenin are associated with arrhythmogenic right ventricular cardiomyopathy

AIMS Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a major cause of juvenile sudden death and is characterized by fibro-fatty replacement of the right ventricle. Mutations in several genes encoding desmosomal proteins have been identified in ARVC. We speculated that αT-catenin, encoded by CTNNA3, might also carry mutations in ARVC patients. Alpha-T-catenin binds plakophilins and this binding contributes to the formation of the area composita, which strengthens cell-cell adhesion in contractile cardiomyocytes. METHODS AND RESULTS We used denaturing high-performance liquid chromatography and direct sequencing to screen CTNNA3 in 76 ARVC patients who did not carry any mutations in the desmosomal genes commonly mutated in ARVC. Mutations c.281T > A (p.V94D) and c.2293_2295delTTG (p.del765L) were identified in two probands. They are located in important domains of αT-catenin. Yeast two-hybrid and cell transfection studies showed that the interaction between the p.V94D mutant protein and β-catenin was affected, whereas the p.del765L mutant protein showed a much stronger dimerization potential and formed aggresomes in HEK293T cells. CONCLUSION These findings might point to a causal relationship between CTNNA3 mutations and ARVC. This first report on the involvement of an area composita gene in ARVC shows that the pathogenesis of this disease extends beyond desmosomes. Since the frequency of CTNNA3 mutations in ARVC patients is not rare, systematic screening for this gene should be considered to improve the clinical management of ARVC families.

A radial glia-specific role of RhoA in double cortex formation

The positioning of neurons in the cerebral cortex is of crucial importance for its function as highlighted by the severe consequences of migrational disorders in patients. Here we show that genetic deletion of the small GTPase RhoA in the developing cerebral cortex results in two migrational disorders: subcortical band heterotopia (SBH), a heterotopic cortex underlying the normotopic cortex, and cobblestone lissencephaly, in which neurons protrude beyond layer I at the pial surface of the brain. Surprisingly, RhoA(-/-) neurons migrated normally when transplanted into wild-type cerebral cortex, whereas the converse was not the case. Alterations in the radial glia scaffold are demonstrated to cause these migrational defects through destabilization of both the actin and the microtubules cytoskeleton. These data not only demonstrate that RhoA is largely dispensable for migration in neurons but also showed that defects in radial glial cells, rather than neurons, can be sufficient to produce SBH.

RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes

RhoA is a small GTPase shown to be crucial for cytokinesis, stress fiber formation, and epithelial cell–cell contacts. Analyzing mice with a keratinocyte-restricted deletion of the RhoA gene, we find that RhoA is not required for skin development and maintenance but has specific functions in vitro.

Expression and Nuclear Location of the Transcriptional Repressor Kaiso Is Regulated by the Tumor Microenvironment

Kaiso is a BTB/POZ zinc finger protein originally described as an interaction partner of p120ctn. In cultured cell lines, Kaiso is found almost exclusively in the nucleus, where it generally acts as a transcriptional repressor. Here, we describe the first in situ immunolocalization studies of Kaiso expression in normal and cancerous tissues. Surprisingly, we found striking differences between its behavior in monolayers of different cell lines, three-dimensional cell culture systems, and in vivo. Although nuclear localization was sometimes observed in tissues, Kaiso was more often found in the cytoplasm, and in some cell types it was absent. In general, Kaiso and p120ctn did not colocalize in the nucleus. To examine this phenomenon more carefully, tumor cells exhibiting strong nuclear Kaiso staining in vitro were injected into nude mice and grown as xenografts. The latter showed a progressive translocation of Kaiso towards the cytoplasm over time, and even complete loss of expression, especially in the center of the tumor nodules. When xenografted tumors were returned to cell culture, Kaiso was re-expressed and was once again found in the nucleus. Translocation of Kaiso to the cytoplasm and down-regulation of its levels were also observed under particular experimental conditions in vitro, such as formation of spheroids and acini. These data strongly imply an unexpected influence of the microenvironment on Kaiso expression and localization. As transcriptional repression is a nuclear event, this phenomenon is likely a crucial factor in the regulation of Kaiso function.

Cytoplasmic redistribution of E-cadherin-catenin adhesion complex is associated with down-regulated tyrosine phosphorylation of E-cadherin in human bronchopulmonary carcinomas

The E-cadherin-catenin complex, by mediating intercellular adhesion, regulates the architectural integrity of epithelia. Down-regulation of its expression is thought to contribute to invasion of carcinoma cells. To investigate the involvement of the E-cadherin-catenin adhesion system in the progression of human bronchopulmonary carcinomas, we compared the immunohistochemical distribution of E-cadherin, alpha-catenin, and beta-catenin in four human bronchial cancer cell lines with different invasive abilities and in 44 primary bronchopulmonary tumors. Although invasive bronchial cell lines did not express E-cadherin and alpha-catenin, complete down-regulation of cadherin-catenin complex expression was a rare event in vivo in bronchopulmonary carcinomas. Nevertheless, a spotty and cytoplasmic pattern of E-cadherin and catenins was observed in 32 primary tumors, only in invasive tumor clusters. Immunoprecipitation experiments showed that this redistribution was not related to a disruption of cadherin-catenin interaction but to down-regulated tyrosine phosphorylation of E-cadherin. We conclude that loss of E-cadherin and/or catenins is not a prominent early event in the invasive progression of human bronchopulmonary carcinomas in vivo. The decreased tyrosine phosphorylation of E-cadherin may reflect a loss of functionality of the complex and implicates a major role in tumor invasion.

Continuous Cell Injury Promotes Hepatic Tumorigenesis in Cdc42-Deficient Mouse Liver

BACKGROUND & AIMS The Rho small guanosine triphosphatase Cdc42 is critical for diverse cellular functions, including regulation of actin organization, cell polarity, intracellular membrane trafficking, transcription, cell-cycle progression, and cell transformation. This implies that Cdc42 might be required for liver function. METHODS Mice in which Cdc42 was ablated in hepatocytes and bile duct cells were generated by Cre-loxP technology. Livers were examined by histologic, immunohistochemical, ultrastructural, and serum analysis to define the effect of loss of Cdc42 on liver structure. RESULTS Mice lacking Cdc42 in their hepatocytes were born at Mendelian ratios. They did not show increased mortality but showed chronic jaundice. They developed hepatomegaly soon after birth, and signs of liver transformation, such as formation of nodules and tumors, became visible macroscopically at age 6 months. Hepatocellular carcinoma was observed 8 months after birth. Tumors grew slowly and lacked expression of nuclear beta-catenin. Lung metastases were observed at the late stage of carcinogenesis. Immunofluorescent examination and electron microscopy revealed severe defects in the liver. At the age of 2 months, the canaliculi between hepatocytes were greatly enlarged, although the tight junctions flanking the canaliculi appeared normal. Regular liver plates were absent. E-cadherin expression pattern and gap junction localization were distorted. Analysis of serum samples indicated cholestasis. CONCLUSIONS We describe a mouse model in which chronic liver disease leads to hepatocarcinogenesis.